Gram negative coccoid bacterium okadaella gastrococcus

ABSTRACT

A new gram negative coccoid bacterium, Okadaella gastrococcus is disclosed. The bacterium was isolated from an animal with a gastrointestinal disorder.

TECHNICAL FIELD

The present invention relates to an isolated bacterium and uses of thebacterium to diagnose gastrointestinal diseases caused by the bacterium.

BACKGROUND ART

Gastrointestinal disease is a common affliction in animals and humans.Although many aetiological agents including viruses, bacteria andprotozoa, have been recognised to cause this disease, there are stillmany clinical cases from which no causative agent can be identified.Furthermore, many cases do not respond to standard medical treatmentregimes suggesting that some unknown agent or agents may be involved.

There have been several new microorganisms identified recently that havebeen shown to cause gastrointestinal disease in animals includinghumans. In 1984, Marshall and Warren (Lancet 1984: 1312-1314) describedCampylobacter-like, spiral. Gram negative bacteria 2.5 μm in length and0.5 μm in diameter in patients with gastritis and peptic ulcers, nowknown as Helicobacter pylori. In 1987, Dent et al (Lancet 1987 ii:96)reported a new Gram negative spiral bacterium, 3.5-7.5 μm in length and0.9 μm in diameter in patients with gastritis and named it asGastrospirillum hominis. This organism is now called Helicobacterhelimanii.

The present inventor has isolated and identified a new bacterium that isimplicated in gastrointestinal disease in humans.

DISCLOSURE OF INVENTION

A new coccoid form of bacterium, distinct from the coccoid form ofHelicobacter pylori, which can colonise the human stomach was identifiedand isolated from a patient with dyspeptic symptoms and found to sufferfrom a diffused gastritis associated with an erosive gastroduodenitisand multiple superficial ulcerations. The bacterium was resistant tousual triple antibiotic therapy composed of tetracycline, metronidazole,and an H₂ antagonist. Symptomatic remission and histological andelectron microscopic resolution of the gastro-duodenalpathologyfollowing a proton pump inhibitor suggests possible role in thepathogenesis of erosive gastro-duodenitis and multiple superficialulcerations. The organism is coccoid form and varying in size 0.2-0.75μm in diameter, most being 0.3-0.5 μm in diameter, with outer membranepilli. The bacterium has been named by the present inventors asOkadaella gastrococcus Gen. nov, Sp. nov.

In a first aspect, the present invention consists in an isolatedbacterium capable of causing gastrointestinal disease in an animal andhaving the characteristics of Okadaella gastrococcus as herein defined.

Preferably, the bacterium has the following characteristics:

Gram negative;

coccoid;

culturable under microaerophilic and anaerobic conditions;

urease-negative under culture for 2 weeks;

catalase-negative;

oxidase-negative;

contains flagella and may contain microfilamentous hair-like pilli;

bacterial colonies do not fluoresce under ultraviolet light (360 nm);

colonies observed as numerous round “dots” in the Warthin-Starry silverstrain; and

non Helicobacter pylori and non Helicobacter helimanii.

A sample of Okadaella gastrococcus was deposited under the provisions ofthe Budapest Treaty with the Australian Government AnalyticalLaboratories (AGAL) on Sep. 22, 1998 and given Accession Number NM98/08610.

In a second aspect, the present invention consists in a method ofdiagnosing gastrointestinal disease in an animal caused by Okadaellagastrococcus, the method comprising identifying the presence of thebacterium according to the first aspect of the present invention in thegastrointestinal tract of, or a clinical specimen from, the animal.

The bacterium may be detected directly by culturing the bacterium from aclinical specimen, identifying the bacterium by microscopy, or may beidentified indirectly by the use of antibodies to the bacterium. Theclinical specimen may be a biopsy, stool specimen, blood sample, or thelike. It will be appreciated that the discovery of the bacterium and itsassociation with gastrointestinal disease will allow its detection byany of the known methods of the art. It will also be appreciated thatmolecular detection methods like polymerase chain reaction (PCR) canalso be used to identify the presence of the bacterium in a clinicalspecimen.

The presence of the bacterium in an animal that does not have clinicalsigns of gastrointestinal disease may also be indicative ofpre-disposition of that animal to gastrointestinal disease. Therefore,the scope of the second aspect of the present invention also includesscreening for the presence of the bacterium in animals as a means ofdetecting animals that may be susceptible to gastrointestinal diseasecaused by this bacterium.

Preferably, the animal is a human.

In a third aspect, the present invention consists in use of thebacterium according to the first aspect of the present invention todevelop diagnostic tools or aids for the detection or diagnosis ofdisease caused by the bacterium. Such diagnostic tools or aids includemolecular probes devised from the genome of the bacterium andantibodies.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers,or steps.

In order that the present invention may be more clearly understood,preferred forms will be described with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a scanning electron micrograph of gastric biopsy specimenshowing the coccoid bacteria in the damaged mucosal layer, with anerythrocyte and leukocyte for size comparison, ×2,640. Bar=10 μm.

FIG. 2 is an enlargement of the scanning micrograph shown in FIG. 1.

FIG. 3 is a negative stain electron micrograph of the coccoid bacteria,×37,500. Barp=1 μm.

FIG. 4 shows transmission electron micrographs of the bacteriumaccording to the present invention.

DESCRIPTION OF THE INVENTION

In 1983 Warren and Marshall described Campylobacter-like, Gram negative,S-shaped bacilli 2.5 μm in length and 0.5 μm in diameter in patientswith gastritis and peptic ulcers, now known as Helicobacter pylori. In1987, Dent et al reported gram negative spiral bacteria, 3.5-7.5 μm inlength and 0.9 μm in diameter in patients with gastritis and named itGastrospirillum hominis, now called Helicobacter helimannii.

There is no published information available regarding the identificationand isolation of other microorganisms from patients with dyspepsia, eventhough dyspepsia is one of the most common medical complaints in theWorld. The present inventors have been attempting to culture, isolateand identify new microorganisms from patients with dyspepsia. A case ofa young Japanese who suffers from an erosive gastro-duodenitis withmultiple superficial ulcerations with unknown aetiology. The presentinventors succeeded to isolate a coccoid form of organism which is notH. pylori from this patient. Chan et al reported that the presence ofcoccoid forms of H. pylori is the dominant feature of adenocarcinoma ofthe stomach. However, international conflicts exist whether the coccoidform of H. pylori is a viable dormant form or nonviable degenerativeform of the curvilinear microorganism. Therefore, it is very importantto distinguish the coccoid form of the microorganism from Helicobacterspecies which could colonise human gastrointestinal tract. Theendoscopic, microbiological, histological and ultrastructural andmolecular biological findings of the new micro-organism is providedbelow.

Case Report

A 20 year old man presented with dyspepsia, indigestion, heartburn andoccasional history of gastro-oesophageal reflux. He complained of pre-and post-prandial pain which was relieved by occasional “burping”. Hedenied abdominal bloating, nausea, or vomiting. He also denied a historyof melena or recent changes in his bowel habit, except that he sufferedfrom diarrhoea 3-4 times/day around the time when he was found to haveduodenitis and multiple duodenal erosion and superficial ulcerations. Hedenied the use of NSAIDs, his smoking habit was 3-10 cigarettes/day anddrinking habit was 375 ml beer per day until this episode. Familyhistory was negative for peptic ulcer disease and gastro-intestinalmalignancy. As his condition did not respond to daily treatment with aH₂ antagonist for a month, and his H. pylori serology test (HEL-pTEST™II, AMRAD) was positive, he underwent a videoscope endoscopicexamination (Olympus GIF XQ 230) and biopsy. As the routine procedure,at least 6-10 biopsy specimens were collected from gastric body andantrum for histological and microbiolgoical examinations. The biopsyspecimens and gastric aspirates were cultured under a microaerophilicand anaerobic conditions at 35° C. The biopsy specimens were tested forurease production in a Christensen's urea broth and examined under amicroscope with Gram stain. Although the histological andmicrobiological examination did not reveal the presence ofHelicobacter-like microorganisms, he was treated with two weeks courseof the triple combination therapy with tetracycline 1.5 g, metronidazole600 mg and ranitidine 300 mg in divided dose daily (causing greater than98% successful eradication of H. pylori). Further follow-up endoscopicexaminations were performed. In all occasions, the endoscopic appearanceof the stomach was a generalised gastritis with numerous gastricerosions and multiple surface ulcerations. An erosive duodenitis andmultiple surface ulcerations were also seen at the same time. In alloccasions, specimens collected from the gastric body and antrum weresubmitted for histological examination in which were all showed anerosive gastritis with focal superficial ulcerations and focallyincreased numbers of neutrophils and chronic inflammatory cells in thelamina propria. The aetiology of the gastro-duodenopathy could not beidentified by the histological examination. Some epithelial regenerationwas seen only in the specimen collected at the time of the thirdendoscopic examination following one month treatment with omeprasole 20mg b.d. Therefore, he was treated with omeprasole 20 b.d for 4 monthsand the fourth endoscopic examination was performed. The endoscopicappearance was hyperaemic erythematous and congested gastric body andantrum and duodenum without gastro-duodenal erosion and ulcerations. Thehistological examination revealed complete resolution of the erosivegastro-duodenitis and multiple superficial ulcerations. The scanningelectron microscopic examination identified some-what enlarged andswollen scattered single coccoid microorganisms in some areas on thesurface of gastric epithelium. There were no electron microscopicallyidentifiable surface erosion nor ulceration in the stomach nor duodenum.Adequately regenerated microvilli were observed in the stomach andduodenum. While examining the Gram stained specimen, the presentinventors noted Gram negative coccoid bacteria and succeeded to culturethe microorganism. The presence of the coccoid organism in the gastricaspirates was also confirmed by the microbiolgoical and electronmicroscopic examinations. The scanning and transmission electronmicroscopic examinations identified abundant coccoid forms of bacteriabeneath the gastric mucous layer, on the gastric epithelial cells andthe clusters of coccoid forms of bacteria in the erosive lesion of thegastric mucosa and in the superficial ulcers. The characteristics of thecultured organism were studied following Gram (G) staining. Haematoxylinand eosin (H&E) stain and Warthin-Starry silver (WSS) stain beforesubjecting to reviewing all the histological specimens. The bacteriawere difficult to see in H&E stained histological sections. When theycould see, the microorganism was recognised as a basophilic clusteredmass. The organism was observed as a numerous round “dots” in the WSS.The bacteria were abundant in the superficial layer of the gastricmucosa, and could be identified in the erosive lesion of the gastricmucosa and gastric pits. The organism was urease-, catalase-, andoxidase-negative. They converted arginine-β-naphthylamine to arginine.This suggested that the organism possessed arginine aminopeptidase. Themature bacterium possessed unique flagella and were surrounded by finemicrofilamentous hair-like pilli. These structures may not be identifiedin all cases. The 16S rRNA sequencing study confirmed that the coccoidform of bacteria were not phylogenically related with the coccid form ofH. pylori but were closer to Haemophilus haemolyticus. DNA comparisonstudies of the coccoid form and H. haemolyticus demonstrated that themicroorganisms are different. The response to factor X and V which arerequired to determine the genus Haemophilus were also tested with theresults being negative for both. The growth conditions and environmentof Haemophilus are not suitable for the coccoid form of the organism.The growth rate was totally different from Haemophilus which growth canbe detected after 24 hours incubation. The coccid organism, however,takes at least 7 days to give any hint of colony formation. The colonyappearance and morphology are also different. The biochemical testresults were totally different from Haemophilus and could not identifiedby the commercially available identification kit, Neisseria/Haemophilusidentification card (biomerieux, Vitek), nor by RapID ANA II system(Innovative Diagnostic Systems) for identification of anaerobicorganism. Chocolate agar was used as the standard for culturingHaemophilus, which is not optimal for the new organism.

Immunoperoxidase stain study was performed on a paraffin block sectionto exclude the possibility of coccoid forms of H. pylori colonisation.There was no H. pylori sera reaction identified.

Discussion

The microaerophilic and anaerobic gram-negative coccoid form of bacteriawhich colonised the human stomach mucosa have not been culturedpreviously, and their association with an erosive gastro-duodenitis andmultiple superficial ulcerations have not been described. The mainreason why the new bacteria could not have been detected previously isprobably due to their size and appearance which resemble to cell debrissuch as cytoplasmic granules in particular expelled chromatins from cellnuclei on inflamed, erosive and ulcerated gastric mucosal cells. Theorganism may have been seen but regarded as oropharygeal contaminants,or superseded by the presence of H. pylori and regarded as commensals.The possibility also exists that the bacteria were misinterpreted andthought as the coccoid form of H. pylori. At first, it was verydifficult to detect the presence of the organism in histologicalspecimens even by very well trained and experienced pathologists untilthe size, nature and characteristics of the isolated organism which werestained with G, H&E and WSS was recognised. Second, it is quite easy todiscard and neglect the cultured colonies as they are very small andabout 0.5-1 mm in diameter after about 1 week incubation. The Gramstained cultured specimen often appears like a Gram negative artefactrather than clear distinct microorganism as they are densely packed andclustered bacteria and appear like “sand”. The other reason could havebeen due to the neglect of gastric microbiology and the concept that thestomach is a sterile organ as stated by Warren when he identifiedS-shaped bacilli patients with gastritis.

It is important to make an accurate diagnosis of the gastro-duodenalpathology if it is associated with bacterial colonisation like H.pylori, H. helimannii, Okadaell infection (Okada et al 1998) or otherbacterial infections; candida (Ralogeropoulos et al 1988),cryptosporidium (Cersosimo et al 1992), as the treatment choice would bedifferent. One may select triple therapy incorporating tetracycline andmetronidazole which achieves a significantly higher successfuleradication of H. pylori than others (Chiba et al 1992). Okadaellagastrococci were resistant to the combination treatment with ranitidine,tetracycline and metronidazole for 2 weeks. Considering thedisappearance of the organism histologically and microbiologicallyfollowing 4 months treatment with omeprazole coincided with histologicaland symptomatic improvement of this patient, the combination therapywith proton pump inhibitors could produce greater efficacy than otherclasses of acid-suppressing agents in the management of an erosivegastro-duodenitis and peptic ulcer disease due to Helicobacter andOkadaella infection.

The organism isolated by the present inventors does not fit into knowngenus Haemophilus, judging from the colony, cell morphology, growthcharacteristics, antibiotic susceptibility, biochemical tests, and DNAanalysis although the new organism has close phylogenic relationshipswith H. haemolyticus. The genus called Okadaella Gen. nov. (Oka.da.-ella: Jap. n. Okada; N. L. masc. n. Okadella, named after Dr T Okadawho identified the bacterium), gastrococcus Sp. nov. (Gr.n. gastr:pertaining to the stomach; Gr. n. coccus: grain) is proposed for thepresent microorganism. Antibodies raised against this organism andperformance of immuno-histo-fluorescence and indirectimmuno-histochemical studies will be useful to show that the bacteriaisolated are identical to the organisms on the surface of the gastricmucosal epithelium. In addition, DNA hybridisation studies with thegenus Haemophilus can be carried out to develop reliable rapid detectingmethods for Okadaella species utilising PCR and reverse transcriptionprobes for known 16S rRNA and DNA sequences of arginine aminopeptidase,for example.

Biochemical Characteristics of Okadaella gastrococcus

Growth medium—Horse blood agar (eg. Colombia agar)

Optimal growth condition: 35-37° C.

Growth condition: Microseropilic and anaerobic

CO₂: 10%, O₂:4 86%, N₂: 85.14%)

Colony size to reach 0.5-1.0 mm: 7-14 days

Colony morphology: Convex, smooth, translucent

Cell body diameter: 0.3-5 μm

Cell body length: 0.2-0.75 μm

Mole % G+C: not determined

X factor requirement: No

Y factor requirement: No

Catalase: negative

Oxidase: negative

Urease: negative

Indole: negative

Ornithine decarboxylase: negative

Haemolysis: negative

Alkaline-phosphatase: not determined

Glucose: negative

Acid production from

L-arabinose: negative

D-galactose: negative

Maltose: negative

Sucrose: negative

Galactosidase(ONPG): negative

(ONPG: o-nitrophenyl-D-galactosidase)

Frucosidase: negative

Glucosidase: negative

Arginine aminopeptidase: Positive

Nitrate reduction: negative

Nitrite reduction: negative

H₂S production: not determined

G-Glutamyl transpeptidase: not determined

Storage at −70° C. with horse red blood cells or horse serum.

(Positive: 90% or more of strains are positive)

(Negative: 10% or less of strains are positive)

References

Noach L. A., Rolf T. M., Mytgat G. N. Electron microscopic study ofassociation between Helicobacter pylori and gastric and duodenal mucosa,J. Clin Pathol 1994:47:699-704

Ogata T. and Araki K. Electron microscopic study of the morphologicalchanges of gastric mucous cell induced by Helicobacter pylori in humangastric ulcers, J. Submicros Pathol T, 28 (2) 255-264, 1996.

Bode G., Malfertheiner P. and Ditschuneit H. Pathogenic implications ofultrastructural findings in Campylobacter pylori related gastroduodenaldisease Scand J Gastroenterol 1998, 23 (suppl 142), 25-39.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as already described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

Biochemical characteristics of Okadaella (O) and Haemophilus (H)organisms Gc, gastrococcus; GE, gastrofilamenti; inf, influenzae; par,parainfluenzae; haem, haemolyticus Characteristics OGc OGf H. inf H. parH. haem HP G. medium H.B.A. H.B.A. C.A. C.A. C.A. H.B.A. Optimal G. temp35-37 35-37 35-37 35-37 35-37 35-37 G condition MA&An MA&An A/FAn A/FAnA/FAn MA&An Colony size to reach 0.5-1.0 mm 7-14 days 7-14 days 24 hrs24 hrs 24 hrs 3-4 days Colony morph. convex convex convex convex convexconvex TL TL G/TL G/Y TL TL smooth smooth smooth smooth smooth smoothCell diameter um 0.3-0.5 0.5 0.3-0.5 0.5 Cell length um 0.2-0.75 >200.5-3.0 2.5-4.0 Mole % G + C (Tm) ? ? 37-44 40.41 39 35-44 Xfactorrequirement No (No) Yes Yes Yes No Vfactor requirement No No Yes No YesNo Catalase − (−) + +/d + + Oxidase − (−) + + + + Urease − (−) +/−+/− + + Indole − (−) +/− − d − Ornithine decarboxy − (−) +/− +/− − ?Hemolysis − (−) − − + − Alkaline phosphatase ? ? + + + + AMP reactionGlucose − (−) + + + ? Acid production from L-Arabinose − (−) − − − ?D-Galactose − (−) + + + Maltose − (−) + + + Sucrose − (−) − + −-Galactosidase (ONPG) − (−) − +/d − -Fucosidase − (−) − − − -Glucosidase− (−) −? − − -Glucosidase − (−) − − − Arginine aminopeptidase + (+) − −− Nitrate reduction − (−) + + + − Nitrate reduction − (−) − + d H₂Sproduction ? ? − + + − +, 90% or more of strains are positive; d, 11-89%of strains are positive. ONPG, o-nitrophenyl- -Dgalactoside.H.B.A.,horse blood agar; CA, chocolate agar. MA&A, microaerophilic andanaerobic condition; A/FAn, aerobic/facultatively anaerobic.

What is claimed is:
 1. An isolated bacterium capable of causinggastrointestinal disease in an animal and having the characteristics ofOkadaella gastrococcus.
 2. The bacterium according to claim 1 beingOkadaella gastrococcus.
 3. The bacterium according to claim 2 havingAccession Number NM 98/08610.
 4. The bacterium according to claim 1having the following characteristics: Gram negative; coccoid; culturableunder microaerophilic and anaerobic conditions; urease-negative underculture for 2 weeks; catalase-negative; oxidase-negative; containsflagella and may contain microfilamentous hair-like pilli; bacterialcolonies do not fluoresce under ultraviolet light (360 nm); and coloniesobserved as numerous round “dots” in the Warthin-Starry silver strain.5. A method of diagnosing gastrointestinal disease in an animal causedby the bacterium Okadaella gastrococcus, the method comprising detectingthe presence of said bacterium in the gastrointestinal tract of, or in aclinical specimen from, the animal.
 6. The method according to claim 5wherein the bacterium is detected directly by culturing the bacteriumfrom a clinical specimen, identifying the bacterium by microscopy; oridentified indirectly by the use of antibodies to the bacterium, oramplification or detection of unique portions of the bacterium genome.7. The method according to claim 6 wherein the clinical specimen is abiopsy, stool specimen, or blood sample.
 8. The method according toclaim 5 wherein the animal is a human.
 9. A diagnostic tool fordetection of disease caused by a bacterium having the characteristics ofOkadaella gastrococcus comprising said bacterium having thecharacteristics of Okadaella gastrococcus.